This proposal examines the very basic cell biological problems of how eukaryotic cells sort proteins within the Golgi body and separate out those protiens destined for the lysosome, and ultimately deliver these proteins to the lysosome. The simple eukaryote yeast will be used as a model system, since the secretory pathway in yeast is strikingly similar to the pathway in higher eukaryotic cells. It seems very likely that the basic cellular functions that facilitate sorting and transport of lysosomal proteins will be conserved across all eukaryotic cells. Yeast offers a unique opportunity to investigate these complex molecular processes by exploiting the crucial genetic advantage available in yeast yet not possible in higher eukaryotic cells. Yeast mutants have been obtained that are defective either in targeting signals of proteins, sorting or transport of proteins to the lysosome-like vacuole of yeast. These mutants will be characterized by the genetic techniques available in yeast. The epistatic relationships between these targeting, sorting and transport mutants will be investigated. The receptor responsible for sorting vacuolar glycoproteins will be identified, isolated and characterized. The structural gene will be cloned and used to generate mutants that will allow us to ascertain the cellular phenotypes of sorting receptor defective yeast cells. These investigations should provide molecular information on the nature of eukaryotic protein recognition signals and how these signals control the intracellular localization of proteins. The diseases Mucolipidosis II and II (pseudo-Hurler polydystrophy) result from genetically-inherited defects in an enzyme responsible for adding the targeting signal to lysosomal enzymes. These defects result in the secretion of soluble lysosomal proteins. the work described in this proposal should increase our understanding of the specificity of the enzyme defective in Mucolipidosis II and III and thus help elucidate the process of targeting lysosomal proteins in general.